A schematic of the clotting cascades is shown in FIG. 15. In the figure the various clotting factors are indicated by their Roman numeral (i.e., factor VII is indicated by VII). The intrinsic cascade (also referred to as the contact pathway of blood coagulation) is initiated when contact is made between blood and certain artificial surfaces. The extrinsic pathway (also referred to as the tissue factor pathway of blood coagulation) is initiated upon vascular injury which leads to exposure of tissue factor (TF) (also identified as factor III). The dotted arrow represents a point of cross-over between the extrinsic and intrinsic pathways. The two pathways converge at the activation of factor X to Xa. Factor Xa has a role in the further activation of factor VII to VIIa. Active factor Xa hydrolyzes and activates prothrombin to thrombin. Thrombin can then activate factors XI, VII and V furthering the cascade. Ultimately, the role of thrombin is to convert fibrinogen to fibrin, which forms clots.
The fibrinolytic system is responsible for the breakdown of fibrin clots through a series of highly regulated enzymatic reactions. In addition to their role in blood hemostasis, the components of the fibrinolytic system have also been implicated in extracellular matrix degradation and cell migration during inflammation, tumor invasion, tissue repair and angiogenesis (reviewed by [4]). A schematic representation of the proteins and interactions involved in fibrinolysis is shown in FIG. 1. Two main phases are involved: plasminogen activation and subsequent fibrin degradation. Plasminogen activation occurs by the action of either of two serine proteases, tissue-type plasminogen activator (tPA) or urokinase-type plasminogen activator (uPA). The enzymatic activities of these plasminogen activators are opposed by the plasminogen activator inhibitor, PAI-1, a member of the serpin (serine protease inhibitor) superfamily. The product of plasminogen activation is the serine protease, plasmin, whose activity is regulated by another serpin, alpha2-antiplasmin (alpha2-AP). Plasmin proteolytically degrades fibrin into soluble fibrin degradation products (FDPs). Recently, a carboxypeptidase inhibitor of fibrinolysis has been described [5]. This inhibitor serves as a link between coagulation and fibrinolysis, as its main physiological activator is thrombin (especially, thrombin bound to thrombomodulin). For this reason, the molecule is termed Thrombin Activatable Fibrinolysis Inhibitor (TAFI). It should be noted that this inhibitor has also been referred to as carboxypeptidase U, carboxypeptidase R, and plasma carboxypeptidase B, since it was discovered by several groups simultaneously. TAFI circulates as a procarboxypeptidase, which is converted to the active carboxypeptidase enzyme (TAFIa) by limited proteolysis.
Tissue factor pathway inhibitor (TFPI) is one of the physiologically important coagulation inhibitors present in blood. When blood coagulation is initiated, TFPI binds very tightly to the active site of coagulation factor Xa (FXa) and inhibits its enzymatic activity. The inhibited TFPI:FXa complex can then bind to the complex of factor VIIa (FVIIa) and tissue factor (TF), resulting in a fully inhibited tetrameolecular complex (TF:FVIIa:TFPI:FXa). This effectively shuts down further initiation of blood clotting, which is thought to be important in limiting the size of blood clots following injury. In vivo, only a small amount of TFPI circulates in the plasma as free TFPI [24]. (There is another pool of TFPI in plasma that is covalently bound to lipoprotein particles, but it is essentially inactive.) In healthy normal persons, the majority of active TFPI is bound to the endothelial cell surface or is present in platelets [24].
As with other clotting proteins, the plasma levels of TFPI can vary from individual to individual. Normal variation in TFPI levels generally has a limited impact on routine clotting assays for two reasons. First, most normal plasma samples contain fairly low levels of active TFPI (1-20 ng/ml). Second, the influence of natural coagulation inhibitors like TFPI on plasma clotting times is lessened when clotting times are short.
Since routine screening tests for global coagulation function (such as the Prothrombin Time (PT) test) are generally designed to clot very rapidly (the range of normal clotting times in the PT assay is typically 10 to 15 seconds), these tests are only minimally influenced by the normal variation in TFPI levels. However, the influence of TFPI on clotting time in vitro can become significant when the plasma sample contains an unusually high level of TFPI, when other deficiencies in clotting function prolong the clot time and allow TFPI to have a larger influence, or in specialized clotting assays.
Patients with thrombotic episodes are typically treated with anticoagulants. Often, these patients are treated initially with heparin by injection, then gradually switched over to oral anticoagulant therapy (coumadin) for long-term control. The anticoagulant status of patients being treated with coumadin is usually monitored with the PT assay, whose results are used to adjust the coumadin dosage. Multiple studies have described the potential variability of PT results during this period of transition between heparin and coumadin therapy. One possible source of interference in clotting assays in samples from patients undergoing heparin therapy is elevated levels of plasma TFPI. This happens because administration of heparin causes release of the pool of endothelial-bound TFPI into the plasma [25]. In patients with increased TFPI activity, prolonged clot times may be misinterpreted as coagulation factor deficiencies or attributed to the effect of oral anticoagulant (coumadin) therapy.
Some studies have demonstrated that elevated levels of plasma TFPI, especially in patients undergoing heparin therapy, can seriously interfere with the proper interpretation of plasma clotting data. This is especially true for the soluble tissue factor-based clotting assay that is used to measuring plasma factor VIIa levels [26,27]. Thus, the anticoagulant effect of elevated TFPI can prolong the observed clotting times in such clotting assays, yielding a falsely depressed measurement of plasma factor VIIa. Inhibitory anti-TFPI antibodies have been successfully used to eliminate interference from elevated levels of TFPI in such assays [26,27]. However, anti-TFPI antibodies are expensive and must be used at high concentrations to completely block TFPI function, which limits their general usefulness. It would therefore be highly desirable to have a simple and inexpensive method for blocking TFPI anticoagulant function in clotting assays.
The Prothrombin Time (PT) test is widely used to monitor oral anticoagulation therapy by coumarins, as a general screening test for the blood clotting system, and as the basis for specific Factor assays. Clotting times obtained with the PT are primarily dependent on the plasma levels of the vitamin K-dependent coagulation Factors II (prothrombin), VII, and X, and on the levels of two non-vitamin K-dependent proteins, Factor V and fibrinogen. Coumarin treatment antagonizes the vitamin K carboxylase/reductase cycle, thus inhibiting the post-translational conversion of glutamate residues to gamma-carboxyglutamate. Vitamin K-dependent clotting factors contain essential gamma-carboxyglutamate residues in their Gla domains. Patients receiving coumarin therapy will therefore produce undercarboxylated vitamin K-dependent clotting factors with reduced procoagulant activity. This prolongs the PT, chiefly due to depression in the levels of Factors II, VII and X. Successful oral anticoagulant therapy with coumarins requires careful monitoring of the patient's PT in order to achieve an effective level of anticoagulation while minimizing bleeding complications (reviewed by Hirsh et al. [1]).
Polyphosphates have been widely used for many years in a number of commercial applications, including uses in food additives, food processing, and water softeners [1]. Polyphosphates have also been used in dentistry in the treatment of periodontal disease, and polyphosphates have been reported to have antimicrobial activity [1]. Polyphosphates have been used to aid in the refolding of recombinant TFPI [19-21].
Inorganic polyphosphate (polyP) is a ubiquitous polymer formed from phosphate residues linked by high-energy phosphoanhydride bonds. PolyP is found in the environment and has been detected in bacterial, fungal, animal and plant cells, although its presence in human cells has remained less studied [1].
In bacteria as well as in several unicellular eukaryotes, such as trypanosomatid and apicomplexan parasites, the green alga Chlamydomonas reinhardtii, and the slime mold Dictyostelium discoideum, polyP is accumulated in acidic organelles known as acidocalcisomes where it can reach millimolar or molar levels [2]. It has recently been found that human platelet dense granules are similar to acidocalcisomes in their density, acidity, and ability to accumulate pyrophosphate (PPi), cations, and polyP [3]. This makes acidocalcisomes the only known class of organelles that has been conserved during evolution from bacteria to humans.